Plants produce and accumulate within their bodies, numerous kinds of low molecular weight organic compounds such as terpenoids, alkaloids, phenolics, and saponins. It was initially thought that these compounds were not directly responsible for the life maintenance of organisms and have only auguxilliary functions; therefore, they were referred to as “secondary metabolites.”
However, in recent years it has been beginning to be understood that these secondary metabolites function as substances responsible for the cell differentiation or the defense against exogenous factors. Concurrently, the methods of utilization are being found in the broad fields of taste products, drugs and pigments. Not to mention the agricultural field, their utility is catching attention in broad fields.
Because such secondary metabolites are valuable in being industrially utilized, the elucidation of their formation process in plant cells has progressed and presently it has been shown that the substances are synthesized through a complex cascade involving a large number of enzymes. Direct Extraction from plants is, however, needed to obtain such substances. In those cases the quantities that can be isolated at one time are very small, resulting in high cost. Therefore, it has been desired that in vitro synthetic methods using gene manipulations or cultured cells be developed.
Farnesyl pyrophosphate synthase is known as an enzyme that is involved in the synthetic cascade of the secondary metabolites in plants. Farnesyl pyrophosphate synthase is an enzyme that is involved in the metabolism of isoprenoids which forms the basis for a variety of substances in plants such as pigments, odorants, phytohormones, phytoalexins, and defense substances against pests (Plant Biochemistry & Molecular Biology, Hans-Walter Heldt, Oxford University Press, pp. 360-376, 1997). It has been shown that farnesyl pyrophosphate synthase catalyzes the reaction converting isopentenyl pyrophosphate into geranyl pyrophosphate by adding dimethylallyl pyrophosphate thereto as well as catalyzes the reaction converting said geranyl pyrophosphate into farnesyl pyrophosphate by adding isopentenyl pyrophosphate thereto.
Hop is a principal material to give beer refreshing bitter taste and flavor. It is beginning to be understood that the secondary metabolites are secreted in large quantities in luplin gland contained in the cone of the hop and these secondary metabolites greatly contribute to the bitter taste and flavor of beer. Further, in recent years it has been shown that the secondary metabolites of the hop possess pharmacological actions (for example, Biosci. Biotech. Biochem., 61 (1), 158, 1997). Under such circumstances, a variety of breeding improvements are being made to the hop with an emphasis on the secondary metabolites accumulated in the luplin gland such as bitter substances and essential oil constituents.
However, the hop is a dioecious plant. Especially, the male plant does not beer cones that serves as the beer material and thus is not regarded important commercially. Very little study, therefore, has been carried out and the genetic traits that will be useful for fermentation have been hardly elucidated. For these reasons, conventional breeding methods through crossing largely rely on experience and intuition. The present situation is that the brewing qualities are totally unpredictable until the cones have grown. Accordingly, it is strongly desired that the farnesyl pyrophosphate synthase gene be isolated from a hop and that the control of the secondary metabolites in the hop and in vitro synthetic methods be established according to an approach using gene manipulations.
The breeding methods utilizing genetic engineering such as transformation techniques and molecular selection techniques are becoming possibilities in various plants. In these methods, more objective and efficient breeding is possible as compared to the conventional breeding methods which largely rely on experience and intuition. More specifically, the transformation technique is one in which an exogenous gene is introduced into a plant cell to have it expressed and the trait to be desirably incorporated is directly introduced into the cell. In order to have the exogenous gene expressed, the following method may be employed: an objective structural gene and a terminator operable in a plant cell are linked to an operable promoter capable of regulating the expression of the gene in the plant cell and the linkage is introduced into the plant cell. For a promoter that is frequently used at the experimental level, there are mentioned, among others, a CaMV 35S promoter, a nopaline synthase gene promoter both of which can express transgenes in relatively large kinds of plants regardless of their tissues (Sanders P. R. et al., Nucleic Acid Res, 15 (1987) 1543-1558). However, when the aforementioned promoters are used to express the transgenes in all the tissues, some transgenes may do harm to the growth of the plants. It is, therefore, strongly desired that tissue-specific promoters capable of expressing exogenous genes in the objective tissue be isolated.